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Decreased expression of vitamin D receptors in neointimal lesions following coronary artery angioplasty in atherosclerotic swine.

Gupta GK, Agrawal T, Del Core MG, Hunter WJ, Agrawal DK - PLoS ONE (2012)

Bottom Line: Interestingly, there was significantly decreased expression of VDR in PCASMCs of neointimal region compared to normal media.Calcitriol has anti-proliferative properties in PCASMCs and these actions are mediated through VDR.This could be a potential mechanism for uncontrolled growth of neointimal cells in injured arteries leading to restenosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences and Center for Clinical and Translational Science, Creighton University School of Medicine, Omaha, Nebraska, United States of America.

ABSTRACT

Background: Inflammatory cytokines, such as TNF-α, play a key role in the pathogenesis of occlusive vascular diseases. Activation of vitamin D receptors (VDR) elicits both growth-inhibitory and anti-inflammatory effects. Here, we investigated the expression of TNF-α and VDR in post-angioplasty coronary artery neointimal lesions of hypercholesterolemic swine and examined the effect of vitamin D deficiency on the development of coronary restenosis. We also examined the effect of calcitriol on cell proliferation and effect of TNF-α on VDR activity and expression in porcine coronary artery smooth muscle cells (PCASMCs) in-vitro.

Methodology/principal findings: Expression of VDR and TNF-α and the effect of vitamin D deficiency in post-angioplasty coronary arteries were analyzed by immunohistochemistry and histomorphometry. Cell proliferation was examined by thymidine and BrdU incorporation assays in cultured PCASMCs. Effect of TNF-α-stimulation on the activity and expression of VDR was analyzed by luciferase assay, immunoblotting and immunocytochemistry. In-vivo, morphometric analysis of the tissues revealed typical lesions with significant neointimal proliferation. Histological evaluation showed expression of smooth muscle α-actin and significantly increased expression of TNF-α in neointimal lesions. Interestingly, there was significantly decreased expression of VDR in PCASMCs of neointimal region compared to normal media. Indeed, post-balloon angioplasty restenosis was significantly higher in vitamin D-deficient hypercholesterolemic swine compared to vitamin D-sufficient group. In-vitro, calcitriol inhibited both serum- and PDGF-BB-induced proliferation in PCASMCs and TNF-α-stimulation significantly decreased the expression and activity of VDR in PCASMCs.

Conclusions/significance: These data suggest that significant downregulation of VDR in proliferating smooth muscle cells in neointimal lesions could be due to atherogenic cytokines, including TNF-α. Vitamin D deficiency potentiates the development of coronary restenosis. Calcitriol has anti-proliferative properties in PCASMCs and these actions are mediated through VDR. This could be a potential mechanism for uncontrolled growth of neointimal cells in injured arteries leading to restenosis.

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Effect of vitamin D deficiency on the development of restenosis following balloon angioplasty.Paraffin embedded thin sections were cut, deparaffinized and stained with H&E and Masson's trichrome stain and immunohistochemical examination was done for proliferating cell nuclear antigen (PCNA). For immunohistochemistry, sections were stained with DAB as chromogen and counterstained using hematoxylin. H&E staining shows the magnitude of neointimal formation in vitamin D-deficient hypercholesterolemic group (A–B) compared to vitamin D-sufficient hypercholesterolemic group (C–D). Masson's trichrome staining show increased smooth muscle cells and extracellular matrix material in neointimal tissue of vitamin D-deficient hypercholesterolemic group (E–F) compared to vitamin D-sufficient hypercholesterolemic group (G–H). Percent area restenosis was calculated by NIH image J as described under “Material and Methods” (I). Expression of PCNA in thin sections of post-balloon angioplasty coronary arteries from vitamin D-deficient hypercholesterolemic (J–K) and vitamin D-sufficient hypercholesterolemic (L–M) swine is shown. Arrows indicate cells expressing PCNA. The bar graph shows the quantitative analysis of the PCNA-positive cells counted in 5 randomly selected fields of the immunostained sections of the post-angioplasty vitamin D-deficient and vitamin D-sufficient swine coronary arteries (N). Data are shown as mean ± SEM. (N = 8) *P<0.5. Scale bar 100 µm, magnification (100×–600×); L: lumen, M: medial layer, NI: neointima.
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pone-0042789-g003: Effect of vitamin D deficiency on the development of restenosis following balloon angioplasty.Paraffin embedded thin sections were cut, deparaffinized and stained with H&E and Masson's trichrome stain and immunohistochemical examination was done for proliferating cell nuclear antigen (PCNA). For immunohistochemistry, sections were stained with DAB as chromogen and counterstained using hematoxylin. H&E staining shows the magnitude of neointimal formation in vitamin D-deficient hypercholesterolemic group (A–B) compared to vitamin D-sufficient hypercholesterolemic group (C–D). Masson's trichrome staining show increased smooth muscle cells and extracellular matrix material in neointimal tissue of vitamin D-deficient hypercholesterolemic group (E–F) compared to vitamin D-sufficient hypercholesterolemic group (G–H). Percent area restenosis was calculated by NIH image J as described under “Material and Methods” (I). Expression of PCNA in thin sections of post-balloon angioplasty coronary arteries from vitamin D-deficient hypercholesterolemic (J–K) and vitamin D-sufficient hypercholesterolemic (L–M) swine is shown. Arrows indicate cells expressing PCNA. The bar graph shows the quantitative analysis of the PCNA-positive cells counted in 5 randomly selected fields of the immunostained sections of the post-angioplasty vitamin D-deficient and vitamin D-sufficient swine coronary arteries (N). Data are shown as mean ± SEM. (N = 8) *P<0.5. Scale bar 100 µm, magnification (100×–600×); L: lumen, M: medial layer, NI: neointima.

Mentions: Balloon angioplasty was performed in the coronary arteries of vitamin D-deficient or vitamin D-sufficient hypercholesterolemic pigs (4 animals per group). After a follow-up of 6-months, animals were euthanized. Development of restenosis was assessed by histomorphometric evaluation. Consistent with our pervious results from coronary injury restenosis model on histomorphometric examination, substantial neointimal formation was observed in porcine coronary arteries following balloon injury, as shown by representative H&E- and Masson's trichrome-stained sections (Fig. 3A–H). Although, the development of neointima was significant in both groups, the percent area restenosis was greater in vitamin D-deficient hypercholesterolemic group (72.48±4.48%) compared to the vitamin D-sufficient hypercholesterolemic group (54.91±3.726) suggesting that vitamin D levels affect the magnitude of restenosis development (Fig. 3I). PCNA-positive cells were increased in balloon-injured vitamin D-deficient hypercholesterolemic coronary arteries compared to vitamin D-sufficient hypercholesterolemic group (Fig. 3J–N).


Decreased expression of vitamin D receptors in neointimal lesions following coronary artery angioplasty in atherosclerotic swine.

Gupta GK, Agrawal T, Del Core MG, Hunter WJ, Agrawal DK - PLoS ONE (2012)

Effect of vitamin D deficiency on the development of restenosis following balloon angioplasty.Paraffin embedded thin sections were cut, deparaffinized and stained with H&E and Masson's trichrome stain and immunohistochemical examination was done for proliferating cell nuclear antigen (PCNA). For immunohistochemistry, sections were stained with DAB as chromogen and counterstained using hematoxylin. H&E staining shows the magnitude of neointimal formation in vitamin D-deficient hypercholesterolemic group (A–B) compared to vitamin D-sufficient hypercholesterolemic group (C–D). Masson's trichrome staining show increased smooth muscle cells and extracellular matrix material in neointimal tissue of vitamin D-deficient hypercholesterolemic group (E–F) compared to vitamin D-sufficient hypercholesterolemic group (G–H). Percent area restenosis was calculated by NIH image J as described under “Material and Methods” (I). Expression of PCNA in thin sections of post-balloon angioplasty coronary arteries from vitamin D-deficient hypercholesterolemic (J–K) and vitamin D-sufficient hypercholesterolemic (L–M) swine is shown. Arrows indicate cells expressing PCNA. The bar graph shows the quantitative analysis of the PCNA-positive cells counted in 5 randomly selected fields of the immunostained sections of the post-angioplasty vitamin D-deficient and vitamin D-sufficient swine coronary arteries (N). Data are shown as mean ± SEM. (N = 8) *P<0.5. Scale bar 100 µm, magnification (100×–600×); L: lumen, M: medial layer, NI: neointima.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3412822&req=5

pone-0042789-g003: Effect of vitamin D deficiency on the development of restenosis following balloon angioplasty.Paraffin embedded thin sections were cut, deparaffinized and stained with H&E and Masson's trichrome stain and immunohistochemical examination was done for proliferating cell nuclear antigen (PCNA). For immunohistochemistry, sections were stained with DAB as chromogen and counterstained using hematoxylin. H&E staining shows the magnitude of neointimal formation in vitamin D-deficient hypercholesterolemic group (A–B) compared to vitamin D-sufficient hypercholesterolemic group (C–D). Masson's trichrome staining show increased smooth muscle cells and extracellular matrix material in neointimal tissue of vitamin D-deficient hypercholesterolemic group (E–F) compared to vitamin D-sufficient hypercholesterolemic group (G–H). Percent area restenosis was calculated by NIH image J as described under “Material and Methods” (I). Expression of PCNA in thin sections of post-balloon angioplasty coronary arteries from vitamin D-deficient hypercholesterolemic (J–K) and vitamin D-sufficient hypercholesterolemic (L–M) swine is shown. Arrows indicate cells expressing PCNA. The bar graph shows the quantitative analysis of the PCNA-positive cells counted in 5 randomly selected fields of the immunostained sections of the post-angioplasty vitamin D-deficient and vitamin D-sufficient swine coronary arteries (N). Data are shown as mean ± SEM. (N = 8) *P<0.5. Scale bar 100 µm, magnification (100×–600×); L: lumen, M: medial layer, NI: neointima.
Mentions: Balloon angioplasty was performed in the coronary arteries of vitamin D-deficient or vitamin D-sufficient hypercholesterolemic pigs (4 animals per group). After a follow-up of 6-months, animals were euthanized. Development of restenosis was assessed by histomorphometric evaluation. Consistent with our pervious results from coronary injury restenosis model on histomorphometric examination, substantial neointimal formation was observed in porcine coronary arteries following balloon injury, as shown by representative H&E- and Masson's trichrome-stained sections (Fig. 3A–H). Although, the development of neointima was significant in both groups, the percent area restenosis was greater in vitamin D-deficient hypercholesterolemic group (72.48±4.48%) compared to the vitamin D-sufficient hypercholesterolemic group (54.91±3.726) suggesting that vitamin D levels affect the magnitude of restenosis development (Fig. 3I). PCNA-positive cells were increased in balloon-injured vitamin D-deficient hypercholesterolemic coronary arteries compared to vitamin D-sufficient hypercholesterolemic group (Fig. 3J–N).

Bottom Line: Interestingly, there was significantly decreased expression of VDR in PCASMCs of neointimal region compared to normal media.Calcitriol has anti-proliferative properties in PCASMCs and these actions are mediated through VDR.This could be a potential mechanism for uncontrolled growth of neointimal cells in injured arteries leading to restenosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences and Center for Clinical and Translational Science, Creighton University School of Medicine, Omaha, Nebraska, United States of America.

ABSTRACT

Background: Inflammatory cytokines, such as TNF-α, play a key role in the pathogenesis of occlusive vascular diseases. Activation of vitamin D receptors (VDR) elicits both growth-inhibitory and anti-inflammatory effects. Here, we investigated the expression of TNF-α and VDR in post-angioplasty coronary artery neointimal lesions of hypercholesterolemic swine and examined the effect of vitamin D deficiency on the development of coronary restenosis. We also examined the effect of calcitriol on cell proliferation and effect of TNF-α on VDR activity and expression in porcine coronary artery smooth muscle cells (PCASMCs) in-vitro.

Methodology/principal findings: Expression of VDR and TNF-α and the effect of vitamin D deficiency in post-angioplasty coronary arteries were analyzed by immunohistochemistry and histomorphometry. Cell proliferation was examined by thymidine and BrdU incorporation assays in cultured PCASMCs. Effect of TNF-α-stimulation on the activity and expression of VDR was analyzed by luciferase assay, immunoblotting and immunocytochemistry. In-vivo, morphometric analysis of the tissues revealed typical lesions with significant neointimal proliferation. Histological evaluation showed expression of smooth muscle α-actin and significantly increased expression of TNF-α in neointimal lesions. Interestingly, there was significantly decreased expression of VDR in PCASMCs of neointimal region compared to normal media. Indeed, post-balloon angioplasty restenosis was significantly higher in vitamin D-deficient hypercholesterolemic swine compared to vitamin D-sufficient group. In-vitro, calcitriol inhibited both serum- and PDGF-BB-induced proliferation in PCASMCs and TNF-α-stimulation significantly decreased the expression and activity of VDR in PCASMCs.

Conclusions/significance: These data suggest that significant downregulation of VDR in proliferating smooth muscle cells in neointimal lesions could be due to atherogenic cytokines, including TNF-α. Vitamin D deficiency potentiates the development of coronary restenosis. Calcitriol has anti-proliferative properties in PCASMCs and these actions are mediated through VDR. This could be a potential mechanism for uncontrolled growth of neointimal cells in injured arteries leading to restenosis.

Show MeSH
Related in: MedlinePlus